Voltage-dependent potassium channels (Kv) are integral membrane proteins that, in response to membrane voltage changes, catalyze potassium ions to diffuse across the cell membrane. Kv channels regulate membrane excitability and are essential to many physiological processes such as the rhythmic beating of heart, the communication between neurons, and the secretion of hormones. The beta subunit (Kv-beta) of the Shaker type Kv channels (Kv1) permanently attaches to the intracellular side of a channel and is implicated in channel modulation during oxidative stresses and hypoxic conditions. Sequence conservation suggests that Kv-beta resembles an aldo-keto reductase (AKR), and the crystal structure of a Kv-beta showed that it has a canonical AKR fold, a tightly bound cofactor nicotinamide adenine dinucleotide phosphate (NADPH), and highly conserved catalytic residues in the right geometry for catalysis to happen. However, the enzymatic activity of Kv-beta has never been demonstrated. The overall objectives of this proposal are to examine how Kv-beta as a functional AKR modulates channel function, to investigate how the enzymatic activity is coupled to channel activities, and to develop an atomic level understanding of the coupling mechanism. The long-term goals of the project are to understand the physiology of Kv-beta, and the principles governing Kv channel modulations. We have recently identified several Kv-beta substrates, and demonstrated that Kv-beta is a functional aldo-keto reductase. We also found that the substrates modulate channel function only when a Kv-beta is co- expressed. These exciting new results led us to hypothesize that: 1) the AKR function of Kv-beta is coupled to Kv channel functions; 2) the coupling is achieved through interactions between intracellular domains and Kv-beta 3) different redox states of Kv-beta have different conformations that induce a conformational change of a channel domain. To test these hypotheses, we propose the following three specific aims: Aim 1: To examine the functional coupling between channel activities and the AKR activity of Kv-beta. Aim 2: To investigate the molecular bases of the coupling mechanism. Aim 3: To investigate the structural bases of the coupling. Results from this project will help us understand modulations of the various Kv channel families, and will help develop therapeutic reagents that target the macromolecule complex.